Transformants producing substance pf1022 derivatives, methods for producing the same, and novel biosynthesis genes

ABSTRACT

An objective of the present invention is to provide a method for producing substance PF1022 derivatives, in particular PF1022-220 and PF1022-260, by a direct fermentation method, and a transformant to be used for this method. According to the present invention, there is provided a transformant producing substance FF1022 derivatives, which can be obtained by introducing a genes involved in a biosynthetic pathway from chorismic acid to p-aminophenylpyruvic acid, including a papA gene encoding 4-amino-4-deoxychorismate synthase. which gene comprises the DNA sequence encoding the amino acid sequence of SEQ ID NO: 2; a papB gene encoding 4-amino-4-deoxyclaismate mutase, which gene comprises the DNA sequence encoding the amino acid sequence of SEQ ID NO: 4; and a papC gene encoding 4-amino-4-deoxyprepbenate dehydrogenase, which gene comprises the DNA sequence encoding the amino acid sequence of SEQ ID NO: 6, into a phenylalanine auxotrophic host induced from an organism that produces a substance PF1022. According to the present invention, there is also provided a process of producing substance PF1022 derivative, comprising steps of culturing the above-mentioned transformant and collecting the substance PF1022 derivatives.

This application is U.S. national stage of International Application No.PCT/JP02/02782 filed Mar. 22, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to transformants producing a substancePF1022 derivative, and to methods for producing the PF1022 derivativeusing the transformants. Furthermore, the present invention relates tonovel genes involved in a biosynthetic pathway from chorismic acid tophenylpyruvic acid.

2. Background Art

Substance PF1022 has anthelmintic activity and its utilization for drugsfor humans and animals is expected. Substance PF1022 is a cyclicdepsipeptide represented by formula (I):

and is known to be manufactured by a fermentation method (JapanesePatent Application Laid-open Publication No. 35796/1991).

Substance PF1022 is a cyclic depsipeptide composed ofL-N-methylleucine[(CH₃)₂CHCH₂CH(NHCH₃)COOH] (H-L-MeLeu-OH), D-lacticacid [CH₃CH(OH)COOH] (H-D-Lac-OH), and D-phenyllactic acid[C₆H₅CH₂CH(OH)COOH] (H-D-PhLac-OH) bonded by ester bonds and amidebonds.

Substance PF1022 can also be represented by formula (II):Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-D-Lac-L-MeLeu-D-PhLac).

As for substance PF1022 derivatives, seven kinds of derivatives, i.e.,PF1022B, PF1022C, PF1022D, PF1022E, PF1022F, PF1022G, and PF1022H, arereported to be manufactured by a fermentation method (Japanese PatentApplication Laid-open Publication No. 170749/1993, Japanese PatentApplication Laid-open Publication No. 184126/1994, WO98/05655). Further,various substance PF1022 derivatives having anthelmintic activity aremanufactured by chemical synthesis (WO94/19334, WO97/11064, JapanesePatent No. 2874342). Among them, a substance PF1022 derivative,PF1022-220, represented by formula (III):

or formula (IV):Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-p-NO₂PhLac-L-MeLeu-D-Lac-L-MeLeu-D-p-NO₂PhLac),wherein D-p-NO₂PhLac represents D-p-nitrophenyllactic acid, and asubstance PF1022 derivative, PF1022-260, represented by formula (V):

or formula (VI): Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-p-NH₂PhLac-L-MeLeu-D-Lac-L-MeLeu-D-p-NH₂PhLac),wherein D-p-NH₂PhLac represents D-p-aminophenyllactic acid, not onlyhave anthelmintic activity by themselves but also serve an extremelyeffective substance as a raw material for synthesizing substance PF1022derivatives having high anthelmintic activity (Japanese Patent No.2874342).

However, PF1022-220 and PF1022-260 could be manufactured only bychemical synthesis. In manufacturing a cyclic depsipeptide having acomplicated cyclic core, such as substance PF1022, a production methodusing fermentation is advantageous in terms of time generally required,labor, cost, and the like and can be easily carried out, as compared toa chemical synthesis method. Accordingly, a process by directfermentation has been in need also for substance PF1022 derivativesrepresented by PF1022-220 and PF1022-260.

The present inventors introduced a gene involved in a biosyntheticpathway from chorismic acid to p-aminophenylpyruvic acid into anorganism producing secondary metabolites in which a benzene ringskeleton is unsubstituted at the para-position with a functional groupcontaining a nitrogen atom, obtained a transformant, and furtherestablished a method of producing secondary metabolites in which abenzene ring skeleton is substituted at the para-position with afunctional group containing a nitrogen atom, by using this transformant(WO01/23542).

SUMMARY OF THE INVENTION

By applying the method described in WO01/23542 to a host producingsubstance PF1022, the present inventors could confirm that the resultingtransformants produced a substance PF1022 derivative, PF1022-268,represented by formula (VII):

or formula (VIII):Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-p-NO₂PhLac-L-MeLeu-D-Lac-L-MeLeu-D-PhLac)and a substance PF1022 derivative, PF1022-269, represented by formula(IX):

or formula (X):Cyclo(L-MeLeu-D-Lac-L-MeLeu-D-p-NH₂PhLac-L-MeLeu-D-Lac-L-MeLeu-D-PhLac),but could not confirm the production of PF1022-220 and PF1022-260.

On the other hand, the present inventors have successfully obtained atransformant that newly produces substance PF1022 derivatives,PF1022-220 and PF1022-260, by inducing a phenylalanine auxotrophicmutant strain from an organism that produces substance PF1022,transforming this mutant strain with a DNA containing genes involved ina biosynthetic pathway from chorismic acid to p-aminophenylpyruvic acid.The present invention is based on these findings.

An objective of the present invention is to provide a method forproducing substance PF1022 derivatives, in particular PF1022-220 andPF1022-260, by a direct fermentation method, and a transformant to beused for this method.

According to the present invention, there is provided a transformantproducing substance PF1022 derivatives, in particular PF1022-220(formula (III)) and PF1022-260 (formula (V)), which is obtainable byintroducing genes involved in a biosynthetic pathway from chorismic acidto p-aminophenylpyruvic acid (biosynthesis gene) into a phenylalanineauxotrophic host induced from an organism that produces substance PF1022represented by formula (I):

According to the present invention, there is also provided a process ofproducing substance PF1022 derivatives, comprising steps of culturingthe abovementioned transformant and collecting the substance PF1022derivatives.

Another objective of the present invention is to provide novel genesinvolved in the biosynthetic pathway from chorismic acid to pheylpyruvicacid.

Novel genes according to the present invention are

a polynucleotide encoding the amino acid sequence of SEQ ID NO: 27 or amodified sequence of SEQ ID NO: 27 having chorismate mutase activity;and

a polynucleotide encoding the amino acid sequence of SEQ ID NO: 38 or amodified sequence of SEQ ID NO: 38 having prephenate dehydrataseactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction map of a DNA fragment isolated fromStreptomyces venezuelae and the position of open reading frames (ORF)thereon.

FIG. 2 shows the construction of plasmid pTrc-papA.

FIG. 3 shows the amino acid analyzer chromatograms used for detectingenzyme activity of a papA gene product.

FIG. 4 shows the construction of plasmid pTrc-papB.

FIG. 5 shows the amino acid analyzer chromatograms used for detectingenzyme activity of a papB gene product.

FIG. 6 shows the construction of plasmid pET-papC1.

FIG. 7 shows the amino acid analyzer chromatograms used for detectingenzyme activity of a papC gene product.

FIG. 8 shows the construction of plasmids pPF260-A3 and pPF260-A4.

FIG. 9 shows the restriction map of a 6-kb HindIII fragment containing aAbp1 gene.

FIG. 10 shows the restriction map of plasmid pABPd.

FIG. 11 shows the construction of plasmid pPF260-B3.

FIG. 12 shows the construction of plasmid pPF260-C3.

FIG. 13 shows the restriction map of the XbaI DNA fragment and thelocation of a chorismate mutase gene.

FIG. 14 shows the construction of plasmid pCMHRV4.

FIG. 15 shows the HPLC chromatograms used for detecting a substancePF1022 derivative, PF1022-220. A is the chromatogram for standardPF1022-220, B is the chromatogram for the sample prepared from thetransformant, and C is the chromatogram for the sample prepared from thetransformant and standard PF1022-220.

FIG. 16 shows the HPLC chromatograms used for detecting a substancePF1022 derivative, PF1022-260. A is the chromatogram for standardPF1022-260, and B is the chromatogram for the sample prepared from thetransformant.

FIG. 17 shows the restriction map of a SacI fragment isolated from thegenomic DNA of the PF1022 strain (FERM BP-2671) and the location of thePDT gene.

FIG. 18 shows the restriction map of plasmid pDPDT.

FIG. 19 shows the HPLC chromatograms used for detecting a substancePF1022 derivative, PF1022-220. A is the chromatogram for the sampleprepared from strain TF-57, and B is the chromatogram for the sampleprepared from strain TF-45.

DETAILED DESCRIPTION OF THE INVENTION

Deposition of Microorganisms

The PF1022 strain was deposited with the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan),dated Jan. 24, 1989. The accession number is FERM BP-2671.

Escherichia coli (JM109) transformed with plasmid pUC118-papA wasdeposited with the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), dated Sep. 17, 1999. Theaccession number is FERM BP-7256.

Escherichia coli (JM109) transformed with plasmid pTrc-papB wasdeposited with the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), dated Sep. 17, 1999. Theaccession number is FERM BP-7257.

Escherichia coli (JM109) transformed with plasmid pET-papC was depositedwith the International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan), dated Sep. 17, 1999. The accessionnumber is FERM BP-7258.

Escherichia coli (JM109) transformed with plasmid pMKD01 was depositedwith the International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan), dated Jul. 12, 1996. The accessionnumber is FERM BP-5974.

Phenylalanine Auxotrophic Host

The phenylalanine auxotrophic host used in the present invention means aphenylalanine auxotrophic mutant strain which is derived from anorganism originally producing substance PF1022 (referred to as“substance PF1022-producing microorganism”) by treating the organism formutation as described later.

An example of preferable phenylalanine auxotrophic host is a mutantstrain of a substance PF1022-producing microorganism which became torequire auxotrophicity to phenylalanine by almost completely lackingenzyme activity involved in the biosynthetic pathway from chorismic acidto aminophenylpyruvic acid, more specifically chorismate mutase and/orprephenate dehydratase activity, or by significantly reducing theseactivity in the parent strain.

The phenylalanine auxotrophic mutant strain can be obtained by treatinga substance PF1022-producing microorganism with UV light or with amutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrousacid and selecting a mutant strain which cannot grow when cultured in aminimum medium but recovers the growth by adding phenylalanine.

The mutant strain can also be obtained by utilizing recombinant DNAtechnology. Namely, a gene encoding chorismate mutase or prephenatedehydratase is isolated from a substance PF1022-producing microorganismand a target gene on the chromosome is disrupted by homologousrecombination using the isolated gene to obtain the mutant strain ofinterest. Gene disruption can be done according to a known method.

The method for disrupting genes using homologous recombination cangenerally be divided into two groups, one-step gene disruption methodand two-step gene disruption method. The following explanation uses achorismate mutase gene as an example.

In a one-step gene disruption method, an insertion type vector or areplacement type vector is used.

As an insertion type vector, a vector comprising an inactivatedchorismate mutase gene and a selectable marker gene to select atransformant is first prepared. The inactivated chorismate mutase genecan be a gene identical to the original chorismate mutase gene, exceptthat a mutation that singly enables inactivation of chorismate mutasegene is introduced into two separate sites. Such an insertion typevector is introduced into a cell to select a transformant in whichhomologous recombination has taken place with the target chorismatemutase gene on the chromosome in the area between the two mutationsites. In such a transformant, two copies of the chorismate mutase geneexist on the chromosome. However, since the mutation is introduced intoone site of each chorismate mutase gene, the chorismate mutase gene onthe chromosome can be inactivated.

As a replacement type vector, a vector containing a chorismate mutasegene in which a selectable marker gene is inserted within the chorismatemutase gene so as to divide the chorismate mutase gene is prepared. Thisreplacement type vector is introduced into a cell to select atransformant in which homologous recombination has taken place in thearea derived from the chorismate mutase gene on both sides of theselectable marker gene. In such a transformant, the chorismate mutasegene on the chromosome can be inactivated since the target chorismatemutase gene is replaced with a gene having an inserted selectable markergene.

On the other hand, in the two-step gene disruption method, in its firststep, a vector comprising a chorismate mutase gene, in which at leastone mutation that singly enables inactivation of the chorismate mutasegene is introduced, and a selectable marker gene is constructed. Thisvector is introduced into a cell to induce homologous recombination witha target chorismate mutase gene on the chromosome upstream of themutation site of the chorismate mutase gene. As a result, the corevector containing the selectable marker gene is placed between the twocopies of the target chorismate mutase gene on the chromosome. Thus, ofthe two copies of the target chorismate mutase gene, one has a mutationand the other has no mutation.

Next, a part of the vector between the two copies of the targetchorismate mutase gene loops out and homologous recombination is inducedagain downstream of the mutation site. As a result, the vectorcontaining the selectable marker gene and one copy of the targetchorismate mutase gene are lost and the chorismate mutase gene on thechromosome is replaced by the target chorismate mutase gene containing amutation, thereby inactivating the chorismate mutase gene on thechromosome. A strain having such recombination can be selected by usingthe loss of the marker gene as an indicator. Here, it is evident that asimilar result can be obtained when homologous recombination is induceddownstream region of the mutation site in the first step and thenhomologous recombination is induced in the upstream region.

Examples of the preferable phenylalanine auxotrophic hosts in thepresent invention include a phenylalanine auxotrophic mutant straininduced from a filamentous fungus strain which belongs toAgonomycetales, preferably a phenylalanine auxotrophic mutant straininduced from Mycelia sterilia, more preferably a phenylalanineauxotrophic mutant strain induced by strain PF1022 which is depositedwith the National Institute of Advanced Industrial Science andTechnology under an accession number of FERM BP-2671. Most preferable isa phenylalanine auxotrophic mutant strain characterized in that thestrain has been induced from Mycelia sterilia and its phenylalaninerequirement is derived from the lack of endogenous chorismate mutaseactivity and/or prephenate dehydratase activity.

Biosynthesis Genes

Examples of enzymes involved in the biosynthetic pathway from chorismicacid to p-aminophenylpyruvic acid include 4-amino-4-deoxychorismatesynthase, 4-amino-4-deoxychorismate mutase, and4-amino-4-deoxyprephenate dehydrogenase (Blanc, V., et al., Mol.Microbiol., 23, 191-202, 1997). The biosynthetic pathway from chorismicacid to p-aminophenylpyruvic acid can be summarized as follows.

4-amino-4-deoxychorismate synthase acts on chorismic acid to produce4-amino-4-deoxychorismic acid; 4-amino-4-deoxychorismate mutase acts onthe resulting 4-amino-4-deoxychorismic acid to produce4-amino-4-deoxyprephenic acid; and 4-amino-4-deoxyprephenatedehydrogenase acts on the resulting 4-amino-4-deoxyprephenic acid toproduce p-aminophenylpyruvic acid.

The term “4-amino-4-deoxychorismate synthase” as used herein means anenzyme that acts on chorismic acid to transform it into4-amino-4-deoxychorismic acid. The 4-amino-4-deoxychorismate synthase isfound in a wide variety of organisms as a part of the biosynthesissystem from chorismic acid to p-aminobenzoic acid. P-Aminobenzoic acidis synthesized from chorismic acid in a two-step reaction. The formerreaction is catalyzed by 4-amino-4-deoxychorismate synthase, and thelatter reaction is catalyzed by 4-amino-4-deoxychorismate lyase (Green,J. M. and Nichols, B. P., J. Biol. Chem., 266, 12971-12975, 1991).

Reported genes encoding 4-amino-4-deoxychorismate synthase include thosederived from Escherichia coli (Kaplan, J. B. and Nichols, B. P., J. Mol.Biol., 168, 451-468, 1983; Goncharoff, P. and Nichols, B. P., J.Bacteriol., 159, 57-62, 1984), Bacillus subtilis (Slock, J. et al., J.Bacteriol., 172, 7211-7226, 1990), Klebsiella pneumoniae (Kaplan, J. B.et al., J. Mol. Biol., 183, 327-340, 1985; Goncharoff, P. and Nichols,B. P., Mol. Biol. Evol., 5, 531-548, 1988), Streptomycespristinaespiralis (Blanc, V. et al., Mol. Microbiol., 23, 191-202,1997), S. venezuelae (Brown, M. P. et al., Microbiology, 142, 1345-1355,1996), and Saccharomyces cerevisiae (Edman, J. C. et al., Yeast, 9,669-675, 1993), and they can be used. Genes encoding the4-amino-4-deoxychorismate synthase, other than those mentioned above,can also be isolated from organisms having 4-amino-4-deoxychorismatesynthase activity using standard techniques and used in the presentinvention.

On the other hand, the 4-amino-4-deoxychorismate synthase can begenerally divided into two groups: one which is composed of twopolypeptides, such as those derived from Escherichia coli, Bacillussubtilis, or Klebsiella pneumoniae, and the other which is composed ofone peptide, such as those from a part of Actinomycetes or Saccharomycescerevisiae. In the present invention, it is preferable to use a geneencoding the 4-amino-4-deoxychorismate synthase consisting of onepolypeptide since a plurality of genes has to be introduced to a host.

In the present invention, an example of the gene encoding the4-amino-4-deoxychorismate synthase is preferably a gene encoding theamino acid sequence of SEQ ID NO: 2 or a modified sequence of SEQ ID NO:2 having 4-amino-4-deoxychorismate synthase activity. More preferably,it is a gene containing the DNA sequence of SEQ ID NO: 1.

In the present invention, “modified sequence” means a sequence havingone or more, for example one to several, modifications selected from thegroup consisting of a substitution, a deletion, an insertion, and anaddition.

In the present invention, whether a modified amino acid sequence has4-amino-4-deoxychorismate synthase activity or not can be evaluated byallowing the protein comprising said amino acid sequence to act on asubstrate and then detecting the reaction product. For example, it canbe evaluated according to the method described later in Example 2.

The term “4-amino-4-deoxychorismate mutase” as used herein means anenzyme that acts on 4-amino-4-deoxychorismic acid to transform it into4-amino-4-deoxyprephenic acid.

The term “4-amino-4-deoxyprephenate dehydrogenase” as used herein meansan enzyme which acts on 4-amino-4-deoxyprephenic acid to transform itinto p-aminophenylpyruvic acid.

A gene encoding 4-amino-4-deoxychorismate mutase and a gene encoding4-amino-4-deoxyprephenate dehydrogenase are obtained from organisms thatcan biosynthesize p-aminophenylpyruvic acid. More specifically, examplesof such organisms include Streptomyces pristinaespiralis that producespristinamycin I; Streptomyces loidens that produces vernamycin B;Nocardia parafinnica and Corynebacterium hydrocarboclastus that producecorynesin; and Streptomyces venezuelae that produces chloramphenicol.

Among these organisms, Streptomyces pristinaespiralis can be used in thepresent invention since genes which presumably encode4-amino-4-deoxychorismate mutase and 4-amino-4-deoxyprephenatedehydrogenase have already been isolated and their nucleotide sequenceshave been determined (V. Blanc et al., Mol. Microbiol., 23, 191-202,1997).

A number of genes encoding chorismate mutase and prephenatedehydrogenase have been already isolated from bacteria, yeasts, plantsand the like, and these genes can be modified by substituting, deletingor adding appropriate amino acids so as to have4-amino-4-deoxychorismate mutase activity and 4-amino-4-deoxyprephenatedehydrogenase activity, based on protein engineering techniques ordirected evolution techniques. Thus, the resulting modified genes canalso be used in the present invention.

In the present invention, an example of the gene encoding the4-amino-4-deoxychorismate mutase is preferably a gene encoding the aminoacid sequence of SEQ ID NO: 4 or a modified sequence of SEQ ID NO: 4having 4-amino-4-deoxychorismate mutase activity, more preferably a genecontaining the DNA sequence of SEQ ID NO: 3.

In the present invention, whether a modified amino acid sequence has4-amino-4-deoxychorismate mutase activity or not can be evaluated byallowing the protein comprising said amino acid sequence to act on asubstrate and then detecting the reaction product, for example,according to the method described later in Example 3.

In the present invention, an example of the gene encoding the4-amino-4-deoxyprephenate dehydrogenase is preferably a gene encodingthe amino acid sequence of SEQ ID NO: 6 or a modified sequence of SEQ IDNO: 6 having 4-amino-4-deoxyprephenate dehydrogenase activity. Morepreferably, it is a gene containing the DNA sequence of SEQ ID NO: 5.

In the present invention, whether a modified amino acid sequence has4-amino-4-deoxyprephenate dehydrogenase activity or not can be evaluatedby allowing the protein comprising said amino acid sequence to act on asubstrate and then detecting the reaction product. For example, it canbe evaluated according to the method described later in Example 4.

Given the amino acid sequences of enzymes involved in the biosynthesisin the present invention, nucleotide sequences encoding the amino acidsequences can be easily determined, and various nucleotide sequencesencoding the amino acid sequences depicted in SEQ ID NO: 2; SEQ ID NO:4, and SEQ ID NO: 6 can be selected.

Accordingly, biosynthesis genes according to the present inventioninclude, in addition to a part or all of the DNA sequences of SEQ ID NO:1, SEQ ID NO: 3, and SEQ ID NO: 5, DNA sequences encoding the same aminoacid sequences and having degenerate codons. Further, they include RNAsequences corresponding to these sequences.

Transformants

A transformant according to the present invention means a hostcomprising genes involved in the biosynthetic pathway from chorismicacid to p-aminophenylpyruvic acid (biosynthesis gene). The gene to beintroduced into the host means a DNA molecule, which is replicable inthe host cell and in which the genes can be expressed, in particular anexpression vector.

A transformed organism can be obtained by introducing a DNA moleculecomprising genes involved in the biosynthetic pathway from chorismicacid to p-aminophenylpyruvic acid into the host. In the presentinvention, when a plurality of biosynthesis enzyme genes is introducedinto the host, each gene can be contained in either the same ordifferent DNA molecules. Further, when the host cell is a bacterium,each gene can be designed to be expressed as a polycistronic mRNA so asto be made into a single DNA molecule.

The expression vector to be used in the present invention can beappropriately selected from viruses, plasmids, cosmid vectors, and thelike taking the kind of the host cell to be used into consideration. Forexample, lambda bacteriophages and pBR and pUC plasmids can be used whenthe host cell is Escherichia coli; pUB plasmids can be used for Bacillussubtilis; and YEp, YRp, YCp, and YIp plasmid vectors can be used foryeasts.

Among the plasmid vectors to be used, at least one vector preferablycontains a selectable marker to select transformants. A drug resistancegene or a gene complementing an auxotrophic mutation can be used as aselectable marker. Preferable examples of the marker genes to be usedfor each host include an ampicillin resistance gene, a kanamycinresistance gene and a tetracycline gene for bacteria; a tryptophanbiosynthesis gene (trp 1), an uracil biosynthesis gene (ura3) and aleucine biosynthesis gene (leu2) for yeasts; and a hygromycin Bresistance gene, a bialaphos resistance gene, a bleomycin resistancegene and an aureobasidin resistance gene for fungi.

Furthermore, in an expression vector, DNA sequences necessary forexpression of the individual genes, for example, transcriptionregulatory signals and translation regulatory signals, such as apromoter, a transcription initiation signal, a ribosome binding site, atranslation stop signal, and a transcription stop signal, can operablybe linked to the biosynthesis gene. The regulatory sequences can beselected and ligated according to an ordinary method.

For example, promoters such a lactose operon and a tryptophan operon canbe used in Escherichia coli; promoters of an alcohol dehydrogenase gene,an acid phosphatase gene, a galactose utilization gene, and aglyceraldehyde 3-phosphate dehydrogenase gene can be used in yeasts; andpromoters such as α-amylase gene, a glucoamylase gene, acellobiohydrolase gene, a glyceraldehyde 3-phosphate dehydrogenase gene,and an Abp1 gene can be used in fungi.

Transformation of a host can be carried out according to an ordinarymethod such as the calcium ion method, the lithium ion method, theelectroporation method, the PEG method, the Agrobacterium method, andthe particle gun method, and the method can be selected depending on thehost to be transformed.

A transformant according to the present invention is preferably atransformant producing a substance PF1022 derivative depicted by formula(III) or formula (V), characterized in that (a) it is derived fromMycelia sterilia producing substance PF1022 depicted by formula (I), (b)an endogenous chrismate mutase gene and/or prephenate dehydratase geneis disrupted by gene disruption, and (c) a gene involved in thebiosynthetic pathway from chorismic acid to p-aminophenylpyruvic acid isintroduced.

Production of Substance PF1022 Derivative

In the present invention, a transformant of the present invention iscultured, and the resultant culture is used to obtain a substance PF1022derivative of interest. The transformant can be cultured according to anordinary method by appropriately selecting a medium, culture conditions,and the like.

The medium can be supplemented with a carbon source and nitrogen sourcethat can be anabolized and utilized, respectively, by the transformantof the present invention, inorganic salts, various vitamins, variousamino acids such as glutamic acid and asparagine, trace nutrients suchas nucleotides, and selectable agents such as antibiotics.

Further, organic and inorganic substances that help the growth of thetransformant of the present invention and promote the production of thesubstance PF1022 derivative of the present invention can beappropriately added. Further, if necessary, a synthetic medium orcomplex medium which appropriately contains other nutrients can be used.

Any kind of carbon source and nitrogen source can be used in the mediumas long as they can be utilized by the transformant of the presentinvention. As the anabolizable carbon source, for example, variouscarbohydrates, such as sucrose, glucose, starch, glycerin, fructose,maltose, mannitol, xylose, galactose, ribose, dextrin, animal and plantoils and the like, or hydrolysates thereof, can be used. The preferableconcentration generally is from 0.1% to 5% of the medium.

As the utilizable nitrogen source, for example, animal or plantcomponents, or exudates or extracts thereof, such as peptone, meatextract, corn steep liquor, and defatted soybean powder, organic acidammonium salts such as succinic acid ammonium salts and tartaric acidammonium salts, urea, and other various inorganic or organicnitrogen-containing compounds can be used.

Further, as inorganic salts, for example, those which can producesodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoricacid, sulfuric acid, and other ions can be appropriately used.

Any medium which contains other components, such as cells, exudates orextracts of microorganisms such as yeasts, and fine plant powders, canbe appropriately used as long as they don't interfere with the growth ofthe transformant and the production and accumulation of the substancePF1022 derivative of the present invention. When a mutant strain havinga nutritional requirement is cultured, a substance to satisfy itsnutritional requirement is added to the medium. However, this kind ofnutrient may not necessarily be added when a medium containing naturalsubstances is used.

The pH of the medium is, for example, about 6 to 8. Incubation can becarried out by a shaking culture method under an aerobic condition, anagitation culture method with aeration or an aerobic submerged culturemethod.

An appropriate incubation temperature is 15° C. to 40° C., generallyabout 26° C. to 37° C.

Production of the substance PF1022 derivative of the present inventiondepends on a medium, culture conditions, or a host used. However, themaximum accumulation can generally be attained in 2 to 25 days by anyculture method. The incubation is terminated when the amount of thesubstance PF1022 derivative of the present invention in the mediumreaches its peak, and the target substance is isolated from the cultureand then purified.

Needless to say, the culture conditions such as the medium component,medium fluidity, incubation temperature, agitation speed and aerationrate can be appropriately selected and controlled depending on thetransformant to be used and the exterior conditions so as to obtainpreferable results. If foaming occurs in a liquid medium, a defoamingagent such as silicone oil, vegetable oils, mineral oils, andsurfactants can be appropriately used.

The substance PF1022 derivative of the present invention accumulated inthe culture thus obtained is contained in the cells of the transformantof the present invention and the culture filtrate. Accordingly, it ispossible to recover the substance PF1022 derivative of the presentinvention from both culture filtrate and transformant cells byseparating the culture into each fraction by centrifugation.

The substance PF1022 derivative of the present invention can berecovered from the culture filtrate according to an ordinary method usedfor recovering the substance PF1022 derivative of the present inventionfrom the culture. The procedures can be carried out singly, incombination in a certain order, or repeatedly. For example, extractionfiltration, centrifugation, salting out, concentration, drying,freezing, adsorption, detaching, means for separation based on thedifference in solubility in various solvents, such as precipitation,crystallization, recrystallization, reverse solution, counter-currentdistribution, and chromatography, can be used.

Further, the substance PF1022 derivative of the present invention can beobtained from the culture inside the cells of the transformant of thepresent invention. For example, extraction from the culture (e.g.,smashing and pressure disruption), recovery (e.g., filtration andcentrifugation), and purification (e.g., salting out and solventprecipitation) can be carried out using an ordinary method.

The crude substance obtained can be purified according to an ordinarymethod, for example, by column chromatography using a carrier such assilica gel and alumina or reverse-phase chromatography using an ODScarrier. A pure substance PF1022 derivative of the present invention canbe obtained from the culture of the transformant of the presentinvention using the abovementioned methods, either singly or inappropriate combination.

Gene for Enzyme Involved in the Biosynthetic Pathway from Chorismic Acidto Phenylpyruvic Acid

It is still another objective of the present invention to provide anovel gene for an enzyme involved in the biosynthetic pathway fromchorismic acid to phenylpyruvic acid. The biosynthetic pathway fromchorismic acid to phenylpyruvic acid can be summarized as follows:Chorismate mutase acts on chorismic acid to produce prephenic acid andprephenate dehydratase acts on the resulting prephenic acid to producephenylpyruvic acid.

A novel gene according to the present invention is a polynucleotideencoding the amino acid sequence of SEQ ID NO: 27 or a modified sequenceof SEQ ID NO: 27 having chorismate mutase activity, more preferably apolynucleotide comprising the DNA sequence of SEQ ID NO: 26.

In the present invention, whether a polynucleotide encodes a modifiedamino acid sequence having chorismate mutase activity or not can beevaluated by introducing the polynucleotide into a host (e.g., E. coli)according to known gene recombination technology for expression,allowing the resulting protein to act on a substrate and then detectingthe reaction product (see Examples 2, 3, 4, and 9).

A novel gene according to the present invention is also a polynucleotideencoding the amino acid sequence of SEQ ID NO: 38 or a modified sequenceof SEQ ID NO: 38 having prephenate dehydratase activity, more preferablya polynucleotide comprising the DNA sequence of SEQ ID NO: 37.

In the present invention, whether a polynucleotide encodes a modifiedamino acid sequence having prephenate dehydratase activity or not can beevaluated by introducing the polynucleotide into a host (e.g., E. coli)using known gene recombination technology for expression, allowing theresulting protein to act on a substrate and then detecting the reactionproduct (see Examples 2, 3, 4, and 17).

In the present invention, given the amino acid sequence of an enzymeinvolved in a biosynthetic pathway from chorismic acid to phenylpyruvicacid, nucleotide sequences encoding the amino acid sequence can beeasily determined, and various nucleotide sequences encoding the aminoacid sequences depicted in SEQ ID NO: 27 and SEQ ID NO: 38 can beselected. Accordingly, genes involved in a biosynthetic pathway fromchorismic acid to phenylpyruvic acid according to the present inventioninclude, in addition to a part or all of the DNA sequences of SEQ ID NO:26 and SEQ ID NO: 37, DNA sequences encoding the same amino acidsequences and having degenerate codons. Further, they include RNAsequences corresponding to these sequences.

EXAMPLE

The present invention is further illustrated by the following examplesthat are not intended as a limitation, and various changes andmodifications fall within the scope of the invention.

Example 1 Isolation of a gene encoding 4-amino-4-deoxychorismatesynthase, a gene encoding 4-amino-4-deoxychorismate mutase, and a geneencoding 4-amino-4-deoxyprephenate dehydrogenase from Streptomycesvenezuelae

(1) Preparation of Probe DNA Fragment

A 50 ml portion of a liquid medium (2% soluble starch, 1% polypeptone,0.3% meat extract, 0.05% potassium dihydrogenphosphate, pH 7.0) wasprepared in a 250-ml Erlenmeyer flask. The ISP5230 strain and 140-5strain of Streptomyces venezuelae were each inoculated into this mediumand cultured at 28° C. for 24 hours. After culturing, the cells wereharvested from the culture by centrifugation, and the chromosome DNA wasprepared from these cells by the method described in GeneticManipulation of Streptomyces, A Laboratory Manual (D. A. Hopwood et al.,The John Innes Foundation, p71-78, 1985).

Next, PCR was carried out using the above-mentioned chromosomal DNA ofthe Streptomyces venezuelae strain ISP5230 as a template andoligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers. The PCRwas carried out with a TaKaRa LA PCR™ kit Ver. 2.1 (Takara Shuzo Co.,Ltd.) and Gene Amp PCR System 2400 (Perkin-Elmer). A reaction solutioncontaining 1 μl of the chromosomal DNA (equivalent to 0.62 μg), 5 μl of10-fold concentrated reaction buffer attached to the kit, 8 μl of a 2.5mM dNTP solution, 0.5 μl each of the above-mentioned primers prepared ata concentration of 100 pmol/μl, 5 μl of dimethyl sulfoxide (Wako PureChemical Industries, Ltd.), 0.5 μl of TaKaRa LA-Taq (2.5 U), and 29.5 μlof sterile water was made up into a total volume of 50 μl. The reactionwas carried out by repeating incubation of 25 cycles of one minute at94° C., one minute at 50° C. and 3 minutes at 72° C., after pretreatmentat 94° C. for 10 minutes. After the reaction, a portion of the reactionsolution was subjected to agarose gel electrophoresis to confirm that aDNA fragment of approximately 2 kbp was specifically amplified. Then,the remaining reaction solution was extracted withphenol:chloroform:isoamyl alcohol (25:24:1) and precipitated withethanol. The precipitate was redissolved in sterile water, and theresulting solution (60 μl) was digested with restriction enzyme BamHI,after which agarose gel electrophoresis was carried out, and a band ofapproximately 2 kbp was isolated according to an ordinary method torecover a DNA fragment.

This DNA fragment was cloned into the BamHI site of plasmid pTrcHis B(Invitrogen). Since the restriction map of the inserted fragment of theresulting plasmid was identical to that of pabAB gene (U21728) reportedby Brown et al. (M. P. Brown et al., Microbiology, 142, 1345-1355,1996), the pabAB gene was considered to be cloned, and the plasmid wasnamed pTH-PAB. The plasmid pTH-PAB was digested with restriction enzymeBamHI, agarose gel electrophoresis was carried out, and an insertionfragment was isolated and recovered to be used as a probe for thescreening of a chromosomal DNA library described below.

(2) Screening of Chromosomal DNA Library and Isolation of Genes

About 10 μg of the chromosomal DNA of the Streptomyces venezuelae 140-5strain was partly digested with restriction enzyme Sau3AI, after whichagarose gel electrophoresis was carried out to isolate and recover DNAfragments of from 10 kbp to 20 kbp.

About 0.5 μg of the DNA fragments of from 10 kbp to 20 kbp thusrecovered and 1 μg of λDASH II previously double-digested withrestriction enzymes BamHI and XhoI were ligated with T4 DNA ligase andthen packaged in vitro using a Gigapack III packaging extract(Stratagene) to construct a chromosomal DNA library. Plaques were formedby infecting Escherichia coli XLI-Blue MRA with this DNA library.

Plaque hybridization was carried out using the DNA fragment ofapproximately 2 kbp isolated in (1) as a probe and an ECL Direct DNA/RNALabeling Detection System (Amersham Pharmacia Biotech) to screen about24000 plaques. Among positive clones thus obtained, ten clones weresubjected to a secondary screening, and the resulting positive cloneswere purified to prepare phage DNAs.

These phage DNAs were digested with restriction enzyme BamHI, andSouthern analysis was carried out, which revealed that the probe washybridized with two kinds of DNA fragments, i.e., fragments ofapproximately 1.8 kbp and approximately 3.4 kbp. Further, restrictionmap analysis of the phage DNAs revealed that these two kinds of DNAfragments were adjoining on the chromosomal DNA.

Next, the entire nucleotide sequences of these two kinds of DNAfragments were determined using a fluorescent DNA sequencer ABI PRISM377 (Perkin-Elmer). As a result of the subsequent open-reading-frame(ORF) search, ORFs I-IV were found as shown in FIG. 1. The amino acidsequences deduced from each of the ORFs were searched for homology withknown amino acid sequences using database, which revealed that ORF I washomologous to p-aminobenzoic acid-synthesizing enzyme, ORF II washomologous to prephenate dehydrogenase, and ORF III was homologous tochorismate mutase. Genes of ORF I, II and III were then named papA, papCand papB, respectively. nucleotide sequence of papA are each shown inSEQ ID The amino acid sequence encoded by papA and the NO: 2 and SEQ IDNO: 1; the amino acid sequence encoded by papB and the nucleotidesequence of papB are each shown in SEQ ID NO: 4 and SEQ ID NO: 3; andthe amino acid sequence encoded by papC and the nucleotide sequence ofpapC are each shown in SEQ ID NO: 6 and SEQ ID NO: 5.

Example 2 Expression of papA Gene in Escherichia coli

In order to obtain the translation region of the papA gene, PCR wascarried out with the phage DNA derived from the positive clone shown inExample 1 as a template and oligonucleotides of SEQ ID NO: 9 and SEQ IDNO: 10 as primers. The PCR was carried out with KOD Dash (Toyobo Co.,Ltd.) as DNA polymerase using the Gene Amp PCR System 9700(Perkin-Elmer). A reaction solution containing 1 μl of phage DNA(equivalent to 1 μg), 5 μl of 10-fold concentrated reaction bufferattached to the enzyme, 5 μl of a 2 mM dNTP solution, 1 μl each of theabove-mentioned primers prepared at a concentration of 100 pmol/μl, 5 μlof dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 1 μl of KODDash, and 31 μl of sterile water was made up into a total volume of 50μl. The reaction was carried out by repeating incubation of 15 cycles of30 seconds at 94° C., 2 seconds at 50° C. and 30 seconds at 72° C.,after pretreatment at 94° C. for 5 minutes. The reaction solution thusobtained was extracted with phenol:chloroform:isoamyl alcohol (25:24:1)and precipitated with ethanol. The precipitate was redissolved insterile water, and the DNA terminals were blunted using a DNA bluntingkit (Takara Shuzo Co., Ltd.). Further, the 5′ end was phosphorylatedusing T4 DNA kinase (Wako Pure Chemical Industries, Ltd.), after whichagarose gel electrophoresis was carried out, a DNA fragment ofapproximately 2 kbp was isolated, recovered, and cloned into the SmaIsite of plasmid pUC118 to obtain plasmid pUC118-papA (FERM BP-7256).

The nucleotide sequence of the inserted fragment of pUC118-papA (FERMBP-7256) was determined using a fluorescent DNA sequencer ABI PRISM 310Genetic Analyzer (Perkin-Elmer). As a result, it was revealed thatcytosine at position 2043 in the nucleotide sequence of SEQ ID NO: 1 wasreplaced by adenine. Since this replacement was believed to be an errorupon amplification of the DNA fragment by PCR and brought no change inthe amino acid sequence to be encoded, the inserted fragment ofpUC118-papA (FERM BP-7256) was used for the following experiment.

pUC118-papA (FERM BP-7256) was introduced into Escherichia coli JM110,and a plasmid was prepared from the resultant transformant using anordinary method. After digesting with restriction enzyme BclI, agarosegel electrophoresis was carried out to isolate and recover a BclI DNAfragment of approximately 2 kbp.

On the other hand, plasmid pTrc99A (Amersham Pharmacia Biotech) wasdigested with restriction enzyme NcoI, and the DNA terminals wereblunted using Mung Bean Nuclease (Wako Pure Chemical Industries, Ltd.).The resultant fragment was further digested with restriction enzyme SmaIand then self-ligated using T4 DNA ligase to obtain plasmid pTrc101.

pTrc101 was digested with restriction enzyme BamHI and treated withalkaline phosphatase (Takara Shuzo Co., Ltd.), after which the resultantfragment was ligated to the above-mentioned 2 kbp BclI DNA fragment. Aplasmid into which the papA gene was inserted in the correct orientationto the promoter contained in pTrc101 was selected and named pTrc-papA.FIG. 2 shows the process of the above-mentioned plasmid construction.

The Escherichia coli JM109 strain carrying pTrc-papA was cultured in anLB liquid medium (1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodiumchloride) supplemented with 100 μg/ml ampicillin, at 37° C. overnight. A1 ml portion of the resultant culture was inoculated into 100 ml of thesame medium, and incubation was carried out at 30° C. for 4 hours, afterwhich 1 ml of 100 mM isopropylthiogalactoside (IPTG) was added, andincubation was further carried out at 30° C. for 3 hours. Afterincubation, cells were recovered from the culture by centrifugation,suspended in 4 ml of buffer solution for cell homogenization (50 mMTris-HCl (pH 8.0), 5 mM EDTA, 10% glycerol) and then homogenized byultrasonic treatment. After homogenization, the supernatant wasrecovered by centrifugation to obtain a cell extract. Further, theEscherichia coli JM109 strain carrying plasmid pTrc101 was treated inthe same manner to prepare another cell extract.

The cell extracts thus prepared were measured for their enzymaticactivity. Namely, 100 μl of the cell extract, 400 μl of distilled water,and 500 μl of a substrate solution [10 mM barium chorismate (Sigma), 10mM glutamine (Wako Pure Chemical Industries, Ltd.), 10 mM magnesiumchloride, 100 mM MOPS (Wako Pure Chemical Industries, Ltd.), pH 7.5]were mixed and reacted at 30° C. for 2 hours. After reaction, a portionof the reaction solution was analyzed using a full automatic amino acidanalyzer JLC-500/V (JEOL, Ltd.).

As shown in FIG. 3, when the cell extract prepared from the Escherichiacoli carrying pTrc-papA was used, a peak was detected on a positionshowing the same retention time with a standard for4-amino-4-deoxychorismic acid synthesized according to the method ofChia-Yu P. Teng et al. (Chia-Yu P. Teng et al., J. Am. Chem. Soc., 107,5008-5009, 1985). On the other hand, the peak on that position was notfound when the cell extract was boiled or when the cell extract preparedfrom the Escherichia coli carrying pTrc101 was used. Thus, the papA genewas verified to encode 4-amino-4-deoxychorismate synthase.

Example 3 Expression of papB Gene in Escherichia coli

In order to obtain the translation region of the papB gene, PCR wascarried out with the phage DNA derived from the positive clone shown inExample 1 as a template and oligonucleotides of SEQ ID NO: 11 and SEQ IDNO: 12 as primers. The PCR was carried out with KOD Dash (Toyobo Co.,Ltd.) as DNA polymerase using Gene Amp PCR System 9700 (Perkin-Elmer). Areaction solution containing 1 μl of phage DNA (equivalent to 1 μg), 5μl of 10-fold concentrated reaction buffer attached to the enzyme, 5 μlof a 2 mM dNTP solution, 1 μl each of the above-mentioned primersprepared at a concentration of 100 pmol/μl, 5 μl of dimethyl sulfoxide(Wako Pure Chemical Industries, Ltd.), 1 μl of KOD Dash and 31 μl ofsterile water was made up into a total volume of 50 μl. The reaction wascarried out by repeating incubation of 15 cycles of 30 seconds at 94°C., 2 seconds at 50° C. and 30 seconds at 72° C., after pretreatment at94° C. for 5 minutes. The reaction solution thus obtained was extractedwith phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated withethanol. The precipitate was redissolved in sterile water and digestedwith restriction enzyme BamHI, after which agarose gel electrophoresiswas carried out, and a DNA fragment of approximately 0.3 kbp wasisolated according to an ordinary method to recover a DNA fragment.

pTrc101 was digested with restriction enzyme BamHI and treated withalkaline phosphatase (Takara Shuzo Co., Ltd.), after which the resultantfragment was ligated to the above-mentioned 0.3-kbp BamHI DNA fragmentusing T4 DNA ligase. A plasmid into which the papB gene was inserted inthe correct orientation to the promoter contained in pTrc101 wasselected and named pTrc-papB (FIG. 4). The nucleotide sequence of theinserted fragment of pTrc-papB (FERM BP-7257) was determined using afluorescent DNA sequencer ABI PRISM 310 Genetic Analyzer (Perkin-Elmer)to verify that the sequence was identical with the nucleotide sequenceof SEQ ID NO: 3.

The Escherichia coli JM109 strain carrying pTrc-papB (FERM BP-7257) wascultured in an LB liquid medium (1% Bacto-tryptone, 0.5% yeast extract,0.5% sodium chloride) supplemented with 100 μg/ml ampicillin, at 37° C.overnight. A 1 ml portion of the resultant culture was inoculated into100 ml of the same medium, and incubation was carried out at 37° C. for2 hours, after which 1 ml of 100 mM isopropylthiogalactoside (IPTG) wasadded, and incubation was further carried out at 37° C. for 5 hours.After incubation, cells were recovered from the culture bycentrifugation, suspended in 4 ml of buffer solution for cellhomogenization (50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 10% glycerol), andthen homogenized by ultrasonic treatment. After homogenization, thesupernatant was recovered by centrifugation to obtain a cell extract.Further, the Escherichia coli JM109 strain carrying plasmid pTrc101 wastreated in the same manner to prepare a cell extract.

The cell extracts thus prepared were measured for their enzymaticactivity. Namely, 50 μl of the cell extract, 200 μl of distilled water,and 250 μl of a substrate solution [2 mg/ml 4-amino-4-deoxychorismicacid, 10 mM magnesium chloride, 100 mM MOPS (Wako Pure ChemicalIndustries, Ltd.), pH 7.5] were mixed and reacted at 30° C. for 1 hour.After reaction, a portion of the reaction solution was analyzed using afull automatic amino acid analyzer JLC-500/V (JEOL, Ltd.).

As shown in FIG. 5, when the cell extract prepared from the Escherichiacoli carrying pTrc-papB (FERM BP-7257) was used, the peak for4-amino-4-deoxychorismic acid declined and the peak for4-amino-4-deoxyprephenic acid was newly detected. A similar result wasobtained when the cell extract boiled for 5 minutes was used.

On the other hand, when the cell extract prepared from the Escherichiacoli carrying pTrc101 was used, there was no change in the peak for4-amino-4-deoxychorismic acid, and the peak for 4-amino-4-deoxyprephenicacid was not detected. Thus, these results revealed that the papB geneencodes 4-amino-4-deoxychorismate mutase and that the4-amino-4-deoxychorismate mutase encoded by the papB gene hadheat-resistant activity which was not lost even after boiling for 5minutes.

Example 4 Expression of papC Gene in Escherichia coli

In order to obtain the translation region of the papC gene, PCR wascarried out using the phage DNA derived from the positive clone shown inExample 1 as a template and oligonucleotides of SEQ ID NO: 13 and SEQ IDNO: 14 as primers. The PCR was carried out with KOD Dash (Toyobo Co.,Ltd.) as DNA polymerase using Gene Amp PCR System 9700 (Perkin-Elmer). Areaction solution containing 1 μl of phage DNA (equivalent to 1 μg), 5μl of 10-fold concentrated reaction buffer attached to the enzyme, 5 μlof a 2 mM dNTP solution, 1 μl each of the above-mentioned primersprepared at a concentration of 100 pmol/μl, 5 μl of dimethyl sulfoxide(Wako Pure Chemical Industries, Ltd.), 1 μl of KOD Dash and 31 μl ofsterile water was made up into a total volume of 50 μl. The reaction wascarried out by repeating incubation of 15 cycles of 30 seconds at 94°C., 2 seconds at 50° C. and 30 seconds at 72° C., after pretreatment at94° C. for 5 minutes. The reaction solution thus obtained was extractedwith phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated withethanol. The precipitate was redissolved in sterile water, and digestedwith restriction enzyme BamHI, after which agarose gel electrophoresiswas carried out, and a DNA fragment of approximately 1 kbp was isolatedaccording to an ordinary method to recover a DNA fragment.

Plasmid pET-11c (Stratagene) was digested with restriction enzyme BamHIand treated with alkaline phosphatase (Takara Shuzo Co., Ltd.), afterwhich the resultant fragment was ligated to the above-mentioned 1 kbpBamHI DNA fragment using T4 DNA ligase. A plasmid into which the papCgene was inserted in the correct orientation to the promoter containedin pET-11c (FERM BP-7258) was selected and named pET-papC.

The nucleotide sequence of the inserted fragment of pET-papC (FERMBP-7258) was determined using a fluorescent DNA sequencer ABI PRISM 310Genetic Analyzer (Perkin-Elmer) to verify that the sequence wasidentical with the nucleotide sequence of SEQ ID NO: 5.

On the other hand, when the papC gene was expressed using pET-papC (FERMBP-7258), evaluation of properties of papC gene products was expected tobe difficult since the vector-derived peptide composed of 14 amino acidswas added to the N-terminal side of the papC gene products. Therefore,pET-papC (FERM BP-7258) was digested with restriction enzyme NdeI, afterwhich plasmid pET-papC1 was obtained by self-ligation using T4 DNAligase. Use of pET-papC1 made it possible to produce papC gene productsby themselves and not as fusion proteins. The above-mentioned plasmidconstruction process is shown in FIG. 6.

The Escherichia coli BL21 (DE3) strain carrying pET-papC1 was culturedin an LB liquid medium (1% Bacto-tryptone, 0.5% yeast extract, 0.5%sodium chloride) supplemented with 100 μg/ml ampicillin, at 37° C.overnight. A 1 ml portion of the resultant culture was inoculated into100 ml of the same medium, and incubation was carried out at 37° C. for2 hours, after which 1 ml of 100 mM isopropylthiogalactoside (IPTG) wasadded, and incubation was further carried out at 37° C. for 5 hours.After incubation, cells were recovered by centrifugation, suspended in 4ml of buffer solution for cell homogenization (50 mM Tris-HCl (pH 8.0),5 mM EDTA, 10% glycerol), and then homogenized by ultrasonic treatment.After homogenization, the supernatant was recovered by centrifugation toobtain a cell extract. Further, the Escherichia coli BL21 (DE3) straincarrying plasmid pET-11c was treated in the same manner to prepare acell extract.

The cell extracts thus prepared were measured for their enzymaticactivity. Namely, 40 μl of the cell extract, 10 μl of the cell extractwhich was prepared from the Escherichia coli carrying pTrc-papB (FERMBP-7257) described in Example 3 and boiled, 190 μl of distilled water,10 μl of a 10 mM NAD solution, and 250 μl of a substrate solution [2mg/ml 4-amino-4-deoxychorismic acid, 10 mM magnesium chloride, 100 mMMOPS (Wako Pure Chemical Industries, Ltd.), pH 7.5] were mixed andreacted at 30° C. for 1 hour. After reaction, a portion of the reactionsolution was analyzed using a full automatic amino acid analyzerJLC-500/V (JEOL, Ltd.).

As shown in FIG. 7, when the cell extract prepared from the Escherichiacoli carrying pET-papC1 was used, the peak for 4-amino-4-deoxychorismicacid declined, and the peak for 4-amino-4-deoxyprephenic acid to begenerated by the papB gene products disappeared. Sincep-aminophenylpyruvic acid cannot be detected by the full automatic aminoacid analyzer JLC-500/V, its synthesis could not directly be confirmed.

However, a peak for p-aminophenylalanine was detected. This wasgenerated probably due to the transfer of an amino group ofp-aminophenylpyruvic acid generated from papC gene products, byEscherichia coli aminotransferase. On the other hand, when the cellextract boiled and the cell extract which was prepared from theEscherichia coli carrying pET-11c were used, there was no change in thepeak for 4-amino-4-deoxyprephenic acid generated from papB geneproducts. Thus, it was revealed that the papC gene coded for4-amino-4-deoxyprephenate dehydrogenase.

Example 5 Construction of Plasmids pPF260-A3 and pPF260-A4 forIntroduction into Phenylalanine Auxotrophic Microorganism

Plasmids pPF260-A3 and pPF260-A4 for expressing the papA gene in aphenylalanine auxotrophic microorganism were constructed as shown inFIG. 8.

An expression vector pABPd for a PF1022-producing microorganism wasconstructed, and then the DNA fragment obtained from plasmid pUC118-papA(FERM BP-7256) described in Example 2 was ligated to this vector toobtain an expression vector. More specifically, the expression vectorwas constructed as described below.

Isolation of Genomic DNA of Substance PF1022-Producing Microorganism

The genomic DNA of the PF1022 strain (FERM BP-2671) was isolatedaccording to the method of Horiuchi et al. (H. Horiuchi et al., J.Bacteriol., 170, 272-278, 1988). More specifically, cells of thesubstance PF1022-producing strain (FERM BP-2671) were cultured for 2days in a seed medium (2.0% soluble starch, 1.0% glucose, 0.5%polypeptone, 0.6% wheat germ, 0.3% yeast extract, 0.2% soybean cake, and0.2% calcium carbonate; pH 7.0 before sterilization; see Example 1 in WO97/00944), and the cells were recovered by centrifugation (3500 rpm, 10minutes).

The cells thus obtained were lyophilized, suspended in a TE solution (10mM Tril-HCl (pH 8.0), 1 mM EDTA), treated in a 3% SDS solution at 60° C.for 30 minutes, and then subjected to a phenol:chloroform:isoamylalcoholextraction (25:24:1) to remove the cell debris. The extract wasprecipitated with ethanol and treated with Ribonuclease A (Sigma) andProteinase K (Wako Pure Chemical Industries, Ltd.), and then the nucleicacid was precipitated with 12% polyethylene glycol 6000. The precipitatewas subjected to TE-saturated phenol extraction and ethanolprecipitation, and the resulting precipitate was dissolved in a TEsolution to obtain the genomic DNA.

Construction of Genome Library of Substance PF1022-ProducingMicroorganism

The genomic DNA derived from the PF1022 strain (FERM BP-2671) preparedas described above was partially digested with Sau3AI. The product wasligated to the BamHI arm of a phage vector, λEMBL3 Cloning Kit(Stratagene) using T4 ligase (Ligation Kit Ver. 2; Takara Shuzo Co.,Ltd.). After ethanol precipitation, the precipitate was dissolved in aTE buffer. The entire ligated mixture was used to infect Escherichiacoli LE392 strain using a Gigapack III Plus Packaging Kit (Stratagene)to form phage plaques. The 1.3×10⁴ (2.6×10⁴ PFU/ml) phage libraryobtained by this method was used for cloning of the Abp1 gene.

Cloning of the Abp1 Gene from the Genomic DNA Derived from SubstancePF1022-Producing Microorganism

A probe to be used was prepared by amplifying the translation region ofthe Abp1 gene by the PCR method. The PCR was carried out using thegenomic DNA prepared from the substance PF1022-producing microorganismas described above as a template and synthetic primers 8-73U and 8-73R,according to a LETS GO PCR kit (SAWADY Technology). The PCR reaction foramplification was conducted by repeating 25 cycles of 30 seconds at 94°C., 30 seconds at 50° C., and 90 seconds at 72° C. DNA sequences of the8-73U and 8-73R are as follows:

-   8-73U: CTCAAACCAGGAACTCTTTC (SEQ ID NO: 15)-   8-73R: GACATGTGGAAACCACATTTTG (SEQ ID NO: 16)

The PCR product thus obtained was labeled using an ECL Direct System(Amersham Pharmacia Biotech). The phage plaque prepared as describedabove was transferred to a Hybond N+ nylon transfer membrane (AmershamPharmacia Biotech), and after alkaline denaturation, the membrane waswashed with 5×SSC(SSC: 15 mM trisodium citrate, 150 mM sodium chloride)and dried to immobilize the DNA. According to the kit protocol,prehybridization (42° C.) was carried out for 1 hour, after which theabove-mentioned labeled probe was added, and hybridization was carriedout at 42° C. for 16 hours. The nylon membrane was washed according tothe kit protocol described above. The washed nylon membrane was immersedfor one minute in a detection solution and then photosensitized on amedical X-ray film (Fuji Photo Film Co., Ltd.) to obtain one positiveclone. Southern blot analysis of this clone showed that a HindIIIfragment of at least 6 kb was identical with the restriction enzymefragment long of the genomic DNA. FIG. 9 shows the restriction map ofthis HindIII fragment. The HindIII fragment was subcloned into pUC119 toobtain pRQHin/119 for use of the following experiment.

Construction of Expression Vector

The promoter region and the terminator region of the Abp1 gene wereamplified by the PCR method using pRQHin/119 as a template. The PCRmethod was carried out using a PCR Super Mix High Fidelity (LifetechOriental Co., Ltd.) with primers ABP-Neco and ABP-Nbam for promoteramplification and ABP-Cbam and ABP-Cxba for terminator amplification.The amplification reaction was conducted by repeating 25 cycles of 30seconds at 94° C., 30 seconds at 50° C. and 90 seconds at 72° C. The DNAsequences of ABP-Neco, ABP-Nbam, ABP-Cbam and ABP-Cxba are as follows:

-   ABP-Neco: GGGGAATTCGTGGGTGGTGATATCATGGC (SEQ ID NO: 17)-   ABP-Nbam: GGGGGATCCTTGATGGGTTTTGGG (SEQ ID NO: 18)-   ABP-Cbam: GGGGGATCCTAAACTCCCATCTATAGC (SEQ ID NO: 19)-   ABP-Cxba: GGGTCTAGACGACTCATTGCAGTGAGTGG (SEQ ID NO: 20)

Each PCR product was purified with a Microspin S-400 column (AmershamPharmacia Biotech) and precipitated with ethanol, after which thepromoter was double-digested with EcoRI and BamHI, the terminator wasdouble-digested with BamHI and XbaI, and the resulting fragments wereligated one by one to pBluescript II KS+ previously digested with thesame enzymes. The product was digested with XbaI, and a destomycinresistance cassette derived from pMKD01 (WO 98/03667, FERM BP-5974) wasinserted to construct pABPd (FIG. 10). pABPd has the promoter andterminator of the Abp1 gene.

An approximately 2 kbp BclI DNA fragment was prepared from plasmidpUC118-papA (FERM BP-7256) described in Example 2. This fragment wasinserted into the BamHI site of the expression vector pABPd forsubstance PF1022-producing microorganism to obtain plasmid pPF260-A.

Next, pPF260-A was double-digested with restriction enzymes PstI andBamHI to prepare a DNA fragment of approximately 1.7 kbp. This fragmentwas subcloned into PstI and BamHI sites of pUC119 to obtain plasmidpUC119-A. Treatment for site-directed mutagenesis was carried out withpUC119-A as a template DNA and the oligonucleotide of SEQ ID NO: 21 as aprimer using a Muta-Gene in vitro Mutagenesis Kit (Bio-Rad) to obtainplasmid pUC119-A1.

Next, pUC119-A1 and pPF260-A were double-digested with restrictionenzymes PstI and BamHI to prepare DNA fragments of approximately 1.7 kbpand approximately 8.6 kbp, and then these fragments were ligated toobtain plasmid pPF260-A2. Further, pPF260-A2 was digested withrestriction enzyme XbaI and then self-ligated using T4 DNA ligase toobtain plasmid pPF260-A3. Next, plasmid pDHBAR (Watanabe, M. et al.,Appl. Environ. Microbiol., 65, 1036-1044 (1999)) was digested withrestriction enzyme XbaI to obtain an approximately 2.5 kbp DNA fragment.This fragment was digested with restriction enzyme XbaI and the obtainedfragment was ligated with plasmid pPF260-A3 treated with a phosphataseto obtain plasmid pPF260-A4.

Example 6 Construction of Plasmid pPF260-B3 for Introduction intoPhenylalanine Auxotrophic Microorganism

Plasmid pPF260-B3 for expressing the papB gene in a phenylalanineauxotrophic microorganism was constructed as shown in FIG. 11.

An approximately 0.3 kbp BamHI DNA fragment was prepared from plasmidpTrc-papB (FERM BP-7257) described in Example 3. This fragment wasinserted into the BamHI site of the expression vector pABPd (Example 5)to obtain plasmid pPF260-B. pPF260-B was digested with restrictionenzyme XbaI and then self-ligated using T4 DNA ligase to obtain plasmidpPF260-B1.

Next, pPF260-B1 was digested with restriction enzyme PstI to prepare aDNA fragment of approximately 0.6 kbp. This fragment was subcloned intothe PstI site of pUC118 in such a manner that the papB gene and thelacZ′ gene aligned in the same direction to obtain plasmid pUC118-B.Treatment for site-directed mutagenesis was carried out with pUC118-B asa template DNA and the oligonucleotide of SEQ ID NO: 22 as a primerusing a Muta-Gene in vitro Mutagenesis Kit (Bio-Rad) to obtain plasmidpUC118-B1.

Next, pUC118-B1 and pPF260-B1 were digested with restriction enzyme PstIto prepare DNA fragments of approximately 0.6 kbp and approximately 8.0kbp, and then these fragments were ligated to obtain plasmid pPF260-B3.

Example 7 Construction of Plasmid pPF260-C3 for Introduction intoPhenylalanine Auxotrophic Microorganism

Plasmid pPF260-C3 for expressing the papC gene in a phenylalanineauxotrophic microorganism was constructed as shown in FIG. 12.

An approximately 1 kbp BamHI DNA fragment was prepared from plasmidpET-papC (FERM BP-7258) described in Example 4. This fragment wasinserted into the BamHI site of the expression vector pABPd (Example 5)to obtain plasmid pPF260-C. pPF260-C was digested with restrictionenzyme XbaI and then self-ligated using T4 DNA ligase to obtain plasmidpPF260-C1.

Next, pPF260-C1 was double-digested with restriction enzymes PstI andSphI to prepare a DNA fragment of approximately 1.7 kbp. This fragmentwas subcloned into the PstI and SphI sites of pUC118 to obtain plasmidpUC118-C. Treatment for site-directed mutagenesis was carried out withpUC118-C as a template DNA and the oligonucleotide of SEQ ID NO: 23 as aprimer using a Muta-Gene in vitro mutagenesis kit (Bio-Rad) to obtainplasmid pUC118-C1.

Next, pUC118-C1 and pPF260-C1 were double-digested with restrictionenzymes PstI and SphI to prepare DNA fragments of approximately 1.7 kbpand approximately 7.6 kbp, and then these fragments were ligated usingT4 DNA ligase to obtain plasmid pPF260-C3.

Example 8 Isolation of Chorismate Mutase Gene Derived from SubstancePF1022-Producing Microorganism

A chorismate mutase gene derived from a substance PF1022-producingmicroorganism was isolated as follows.

Amplification of Partial Gene Fragment by PCR

Amino acid sequences of chorismate mutase derived from Arabidopsisthariana and Saccharomyces cerevisiae were compared to search for highlyhomologous parts. As a result, since amino acid residues 159-164 and244-249 of the amino acid sequence of chorismate mutase derived fromSaccharomyces cerevisiae were highly homologous, oligonucleotidesextrapolated from these sequences were synthesized. They are shownbelow.

-   CMUC-U: CAYTWYGGNAARTTYGT (SEQ ID NO: 24)-   CMUD-L: TAYTCNACYTSNACYTC (SEQ ID NO: 25)-   (N: A, G, C or T, R: A or G, S: G or C, W: A or T, Y: C or T)

Here in the synthesis, inosine was used for base 9 in CMUC-U and base 6in CMUD-L. Next, cDNA to be used as a template in PCR was prepared asfollows.

The PF1022 strain (FERM BP-2671) was cultured in the medium and underthe conditions described in Example 5 and the resulting cells wererecovered by centrifugation (3000 rpm, 10 minutes). The cells werewashed with purified water, frozen at −80° C. and then disrupted with ablender (Nippon Seiki, AM-3) in the presence of liquid nitrogen. Theresulting product was suspended in a denaturation solution (4 Mguanidine thiocyanate, 25 mM trisodium citrate, 0.5% N-lauryl sarcocinesodium salt, 0.1 M mercaptoethanol) and the suspension was stirred atroom temperature for 5 minutes, neutralized with 2 M sodium acetate (pH4.5), and further stirred, adding TE-saturated phenol. Here,chloroform-isoamyl alcohol (24:1) was added and the admixture wasstirred and then centrifuged to isolate the cell component denaturedwith phenol. The upper layer (water layer) was recovered and nucleicacid was precipitated with isopropanol.

This precipitate was dissolved in a TE buffer solution (10 mM Tris-HCl(pH 8.0), 1 mM EDTA) to make a nucleic acid concentration of 1 mg/ml,and precipitated with 2.5 M lithium chloride (5° C., 2 hours). Theprecipitate was recovered by centrifugation, washed with 70% ethanol,and then redissolved in the TE buffer solution to obtain a total RNAfraction. mRNA was purified from the total RNA fraction using an mRNApurification kit (Amersham Pharmacia Biotech). Further, cDNA wassynthesized with this mRNA as a template using a Time Saver cDNAsynthesis kit (Amersham Pharmacia Biotech).

PCR was carried out with the cDNA of the PF1022 strain (FERM BP-2671) asa template using a SuperTaq premix kit (Sawaday Technology). The PCR,touch down PCR, was performed by repeating 7 cycles of 1 minute at 94°C., 2 minutes at 50° C., and 2 minutes at 72° C. after heat denaturationtreatment at 94° C. for 1 minute, then gradually decreasing annealingtemperature each time by 3° C. up to a total of 28 cycles.

As a result, a fragment of approximately 270 bp was amplified. Thisfragment was subjected to agarose gel electrophoresis and a fragment ofinterest was purified using a Sephagrass band prep kit (PharmaciaBiotech). This fragment was ligated to a pT7-blue T vector (Novagen).The resulting sequence was analyzed using an Auto read sequencing kitand an ALF DNA sequencer II (Pharmacia Biotech). The result showed thatthis PCR fragment contained amino acid residues 171-251 of the sequenceof SEQ ID NO: 27.

Cloning of Chorismate Mutase Gene by Plaque Hybridization

The chromosomal DNA library of the PF1022 strain (FERM BP-2671)described in Example 5 was transferred onto a HyBond-N+ (Amersham) andplaque hybridization was carried out using the abovementioned PCRfragment of approximately 270 bp as a probe according to a DIC system(Behringer Mannheim). As a result, 6 kinds of positive clones wereobtained. Of these 6 kinds of positive clone, an XbaI fragment ofapproximately 7 kbp showing the same length of restriction enzymefragment as Southern blot analysis for chromosomal DNA of the PF1022strain (FERM BP-2671) was cloned into pUC18 to obtain plasmid pCM-Xba.

Nucleotide sequence analysis was carried out with this plasmid using anABIPRISM 377 sequencer (Applied Biosystems). FIG. 13 shows therestriction map of the XbaI fragment and the location of the chorismatemutase gene. The presence of one intron in the chorismate mutase genewas inferred from the nucleotide sequence analysis, and its presence wasconfirmed by cDNA analysis. The nucleotide sequence of the chorismatemutase gene and the amino acid sequence deduced from the nucleotidesequence were shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

Example 9 Disruption of Chorismate Mutase Gene of SubstancePF1022-Producing Microorganism

A plasmid for disrupting the chorismate mutase was prepared as shown inFIG. 14. Plasmid pABPd (FIG. 10) described in Example 5 was digestedwith restriction enzyme XbaI to prepare a DNA fragment of approximately3 kbp containing a destomycin resistance gene. This DNA fragment wasblunted by treating with Mung Bean Nuclease (Nippon Gene). Next, plasmidpCM-Xba described in Example 8 was digested with restriction enzymeHpaI, treated with phosphatase, and then ligated to the abovementionedblunted DNA fragment to construct plasmid pCMHRV4.

Plasmid pCMHRV4 was digested with restriction enzyme XbaI and thensubjected to agarose gel electrophoresis to extract and purify a DNAfragment of approximately 10 kbp. This DNA was then dissolved in a TEbuffer solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to make aconcentration of 1 μg/ml. This DNA solution was used for the followingtransformation experiment.

The substance PF1022-producing microorganism was transformed accordingto the method described in Example 1 of WO97/00944. More specifically,strain PF1022 (FERM BP-2671) was cultured at 26° C. for 48 hours in theseed medium described in Example 5. After cultivation, the resultingmycelia were collected by centrifugation (3000 rpm, 10 minutes) andwashed with a 0.5 M sucrose solution. The mycelia thus obtained weresubjected to protoplast generation by shaking in a 0.5 M sucrosesolution containing 3 mg/ml β-glucuronidase (Sigma), 1 mg/ml chitinase(Sigma) and 1 mg/ml zymolyase (Seikagaku Kogyo) at 30° C. for 2 hours.The mixture thus obtained was filtered to remove the cell debris. Theprotoplasts were washed twice by centrifugation (2500 rpm, 10 minutes,4° C.) with an SUTC buffer solution (0.5 M sucrose, 10 mM Tris-HCl (pH7.5), 10 mM calcium chloride), and then a 1×10⁷/ml protoplast suspensionwas prepared with the SUTC buffer solution.

The previously prepared DNA solution was added to 100 μl of theprotoplast suspension, and the resulting mixture was allowed to standunder ice-cooling for 5 minutes. Then, 400 μl of a polyethylene glycolsolution (60% polyethylene glycol 4000 (Wako Pure Chemical Industries,Ltd.), 10 mM Tris-HCl (pH 7.5), 10 mM calcium chloride) was added tothis mixture, and the resulting admixture was allowed to stand underice-cooling for 20 minutes.

The protoplasts treated as described above were washed with the SUTCbuffer solution and resuspended in the same buffer solution. Theresulting suspension was double-layered onto a potato dextrose agarmedium containing 25 μg/ml hygromycin B and 0.5 M sucrose, together witha potato dextrose soft agar medium. Incubation was carried out at 26° C.for 5 days, and colonies appeared were deemed to be transformants.

When the resulting transformants were seeded on a minimal medium (0.5%glucose, 0.67% yeast nitrogen base w/o amino acids (Difco), 0.12% sodiumglutamate, 0.14% asparagine, 2 μg/ml coline chloride, 1.5% purified agar(Sigma)), there was a strain which did not grow (strain V4M-11). On theother hand, when the strain V4M-11 was seeded on the minimal mediumsupplemented with 50 μg/ml phenylalanine, the growth recovery wasobserved. Accordingly, the strain V4M-11 was revealed to be auxotrophicto phenylalanine.

Next, chorismate mutase activity of the strain V4M-11 was measured asfollows. The parent strain and the strain V4M-11 were cultured under thesame conditions as described in Example 5, after which cells werecollected by centrifugation and suspended in a buffer solution for celldisruption (50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mM DTT, 1 mM PMSF, 10%glycerol). This suspension was treated with ultrasonication and thesupernatant was recovered by centrifugation to obtain a cell extract. 30μl of the cell extract, 20 μl of 1 M Tris-HCl (pH 8.0), and 50 μl of 2mM barium chorismate (Sigma) were mixed and held at 30° C. for 1 hour.

Next, 100 μl of 1 N hydrochloric acid were added and the admixture washeld at 30° C. for 15 minutes, after which 800 μl of 1 N sodiumhydroxide solution were added and the optical density of the solutionwas measured at 320 nm. A sample in which 2 mM barium chorismate wasadded after adding 1 N hydrochloric acid was used as a control. Theresult is shown in Table 1 below.

TABLE 1 A320 Strain After reaction Control ΔA320 Parent strain 0.3630.203 0.160 V4M-11 0.190 0.203 −0.013

The result above revealed that the strain V4M-11 lacked chorismatemutase activity.

Example 10 Transformation of Chorismate Mutase Gene-Disrupted Strain ofPF1022-Producing Microorganism (Phenylalanine Auxotrophic Host)

A mixture of 1 μg of pPF260-A4, 3 μg of pPF260-A3, 3 μg of pPF260-B3,and 3 μg of pPF260-C3 was precipitated with ethanol and then redissolvedin 10 μl of a TE buffer solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA).The DNA solution thus prepared was used to transform the strain V4M-11according to the method described in Example 9, except that 50 μg/mlbialaphos was added instead of hygromycin B.

Chromosomal DNAs were obtained from the resulting transformants, and PCRwas carried out using them as a template DNA under the same conditionsas described in Examples 2, 3 and 4, except that 25 cycles wererepeated, to detect the papa, papB and papC genes. As a result, strainTF-57 was selected as a transformant into which all of the three geneswere introduced.

Example 11 Cultivation of Transformant and Detection of PF1022-220

The transformant strain TF-57 selected in Example 10 was cultured asdescribed in WO 97/20945. Namely, cells were cultured at 26° C. for 2days in the seed medium described in Example 5. A 2 ml portion of eachresultant culture was inoculated into 50 ml of a production medium (0.6%wheat germ, 1.0% pharma media, 2.6% soluble starch, 6.0% starch syrup,0.2% MgSO₄.7H₂O, 0.2% NaCl), and incubation was further carried out at26° C. for 6 days. After incubation, the resulting cells were collectedfrom a 40 ml portion of the culture by centrifugation and then extractedwith 30 ml of ethyl acetate. The extract was concentrated by drying andredissolved in 4 ml of methanol. A 10 μl portion of the solution wassubjected to HPLC analysis.

Conditions for HPLC analysis were as follows:

-   -   HPLC system—LC-10ADVP, Shimadzu Corp.    -   Column—Inertsil ODS-2, 4.6×250 mm    -   Mobile phase—Acetonitorile:water=70:30    -   Flow rate—1.0 ml/min    -   Column temperature—40° C.    -   Detector—UV visible detector SPD-M10AVP, Shimadzu Corp.    -   UV wavelength—272 nm

As shown in FIG. 15, the extract from the transformant strain TF-57exhibited the peak in the same retention time (20.520 min) with standardPF1022-220 (20.308 min). Further, HPLC analysis using a mixture of theextract derived from the transformant and the standard verified that thepeaks derived from the extract and the standard perfectly matched(20.470 min). Measurements of mass spectra for the substances containedin the peak using an LC-MS system (a quadrapole-type bench top LC/MSsystem NAVIGATOR with aQa™, Thermoquest) agreed with that for thestandard. From the result above, it was revealed that the transformantstrain TF-57 produced a substance PF1022 derivative, PF1022-220.

Example 12 Cultivation of Transformant and Detection of PF1022-260

The transformant strain TF-57 selected in Example 10 was cultured underthe conditions as described in Example 11. After incubation, theresulting cells were collected from a 500 ml portion of the culture bycentrifugation and then extracted with 500 ml of methanol. The extractwas concentrated by drying and redissolved in 2 ml of methanol. A 10 μlportion of the solution was subjected to HPLC analysis.

Conditions for HPLC analysis were as follows:

-   -   HPLC system—LC-10ADVP, Shimadzu Corp.    -   Column—Inertsil ODS-2, 4.6×250 mm    -   Mobile phase—Acetonitorile:water=55:45    -   Flow rate—1.0 ml/min    -   Column temperature—40° C.    -   Detector—UV visible detector SPD-M10AVP, Shimadzu Corp.    -   UV wavelength—245 nm

As shown in FIG. 16, the extract from the transformant strain TF-57exhibited the peak in the same retention time (27.301 min) with standardPF1022-260 (27.337 min). Measurements of mass spectra using an LC-MSsystem (a quadrapole-type bench top LC/MS system NAVIGATOR with aQa™,Thermoquest) for the substances contained in the peak agreed with thatfor the standard. From the result above, it was revealed that thetransformant strain TF-57 produced a substance PF1022 derivative,PF1022-260.

Example 13 Partial Purification of Prephenate Dehydratase (PDT)

The PF1022 strain (FERM BP-2671) was cultured in the medium and underthe conditions described in Example 11, after which the cells wererecovered from about 700 ml of the resulting culture fluid bycentrifugation (9,000×g, 30 minutes), the precipitate was suspended in abuffer solution for disruption (50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mMDTT, 1 mM PMSF, 20% glycerol) to wash the cells. Further, centrifuge wascarried out (9000×g, 30 minutes) and the resulting precipitate wasresuspended in 800 ml of the abovementioned buffer solution fordisruption to obtain a cell suspension.

The cell suspension was treated with ultrasonication (10 minutes, 3times) and then centrifuged (9,000×g, 30 minutes, 2 times) to removecell debris and a cell extract was obtained. This cell extract wassubjected to Q Sepharose Fast Flow column chromatography (AmershamPharmacia Biotech, 2.6×32 cm). The flow rate was 1 ml/min. The columnwas then washed with 540 ml of a buffer solution A (50 mM Tris-HCl (pH8.0), 1 mM DTT, 20% glycerol). Further, proteins were eluted with alinear concentration gradient of 0-1.0 M sodium chloride using a totalvolume of 730 ml of the buffer solution A containing 1 M sodiumchloride. Fractions of 20 ml were collected starting 300 minutes afterthe start of the concentration gradient, and each fraction was measuredfor PDT activity as follows: Namely, 20 μl of 2 mM barium prephenate(Sigma), 8 μl of 1 M Tris-HCl (pH 7.0), and 12 μl of an enzyme samplewere mixed and held at 30° C. for 30 minutes, after which 360 μl of 1 Nsodium hydroxide were added and the optical density was measured at 320nm. As a result, high activity was detected in fractions 13-16 and thesefractions were pooled to make a Q Sepharose fraction (80 ml).

Next, 19.36 g of ammonium sulfate were added to the whole volume of theQ Sepharose fraction, centrifugation was carried out (20,000×g, 15minutes), and the resulting precipitate was removed to obtainsupernatant. The supernatant was subjected to Butyl-Toyopearl 650Scolumn chromatography (Toso, 1.6×25 cm). The flow rate was 1 ml/min. Thecolumn was washed with the buffer solution A containing 1.6 M ammoniumsulfate, after which proteins were eluted with a linear concentrationgradient of 1.6-0M ammonium sulfate using a total volume of 100 ml ofthe buffer solution A, and further with 50 ml of the buffer solution A.Fractions of 5 ml were collected starting 50 minutes after the start ofthe concentration gradient, and each fraction was measured for PDTactivity. As a result, activity was detected in fractions 8-15 and thesefractions were pooled to make a Butyl-Toyopearl fraction (40 ml).

13.48 g of ammonium sulfate were added to the whole volume of theButyl-Toyopearl fraction and the resulting precipitate was recovered bycentrifugation (20,000×g, 15 minutes). The precipitate was dissolved in1 ml of a buffer solution B (50 mM sodium phosphate (pH 7.0), 1 mM DTT,20% glycerol), subjected to a HiLoad 26/60 Superdex 200 pg (AmershamPharmacia Biotech, 2.6×60 cm), and eluted using the buffer solution B.The flow rate was 1 ml/min. Fractions of 20 ml were collected over aperiod from 160 minutes to 220 minutes after charging the sample, eachfraction was measured for PDT activity. As a result, activity wasdetected in fractions 4-8 and these fractions were pooled to make aSuperdex 200 fraction (25 ml).

The whole volume of the Superdex 200 fraction was subjected toMacro-Prep Hydroxyapatite column chromatography (Bio-Rad, 0.5×20 cm) andeluted using the buffer solution B. The flow rate was 0.5 ml/min.Fractions of 2 ml were collected after charging the sample and eachfraction was measured for PDT activity. As a result, activity wasdetected in fractions 2-25 and these fractions were pooled to make aHydroxyapatite fraction (38 ml).

The whole volume of the Hydroxyapatite fraction was concentrated using aCENTRICON PLUS-20 (Millipore) and the resulting concentrate was appliedonto a HiTrap Blue HP column (Amersham Pharmacia Biotech, 5 ml) andeluted with the buffer solution B. The flow rate was 1 ml/min. Fractionsof 2 ml were collected after charging the sample and each fraction wasmeasured for PDT activity. As a result, activity was detected infractions 6-22 and these fractions were pooled to make a HiTrap Bluefraction (34 ml).

The whole volume of the HiTrap Blue fraction was concentrated using aCENTRICON PLUS-20 and a Microcon-10 (Amicon) and the resultingconcentrate was applied onto a Superdex 75 HR column (Amersham PharmaciaBiotech, 1.0×30 cm) and then eluted with the buffer solution B. The flowrate was 0.25 ml/min. Fractions of 0.5 ml were collected starting 20minutes after charging the sample and each fraction was measured for PDTactivity. As a result, activity was detected in fractions 7-11.Accordingly, fractions 5-13 were analyzed using SDS-PAGE. Since theintensity of the resulting band of about 35 kDa agreed with theintensity of the enzyme activity, the protein obtained was identified asPDT.

Example 14 Determination of Partial Amino Acid Sequence of PDT

(1) N-Terminal Amino Acid Sequence

The active fraction obtained in Example 13 was subjected to SDS-PAGE(TEFCO) and the protein was electrically transferred onto a PVDFmembrane (Immobilon-PSQ, Millipore) using a Multifor II (AmershamPharmacia Biotech). The membrane was stained with Coomassie BrilliantBlue G250 (Nakarai Tesque), washed with water and dried in air. A bandhaving a molecular weight of about 35 kDa was cut out from thismembrane, and subjected to analysis of the N-terminal amino acidsequence using a protein sequencer Model 492 (Applied Biosystems). As aresult, the following sequence was obtained. X shows an unidentifiedamino acid.

-   N-terminal amino acid sequence: GHTSAGDAGSKPVVXFLGPISSY (23    residues) (SEQ ID NO: 28).    (2) Analysis of Internal Amino Acid Sequence (Peptide Mapping)

The active fraction purified in Example 13 was subjected to SDS-PAGE andstained with Coomassie Brilliant Blue R250 (Nakarai Tesque), and a bandof about 35 kDa was cut out, completely destained at 30° C. in a 0.2 Mammonium bicarbonate buffer solution (pH 8.0) prepared in 50%acetonitrile, and dried in air at room temperature for 2 hours. Next,the resulting shrunken gel piece was moistened with a 0.2 M ammoniumbicarbonate buffer solution (pH 8.0) containing 0.02% Tween 20 and thena 1/50 molar volume of trypsin (Promega) to the protein was added. Thegel piece was allowed to stand at 37° C. for 10 minutes and thenimmersed in the abovementioned buffer solution, after which the reactionwas carried out at 37° C. for 2 days. The supernatant after the reactionwas recovered, and the decomposed product was further recovered from thegel piece with 60% acetonitrile and 0.1% trifluoroacetic acid andcombined with the reaction supernatant. The resulting sample wasconcentrated and then subjected to column chromatography (RP-300Aquapore C18, 220×2.1 mm, concentration gradient: 0.1% trifluoroaceticacid, 5% acetonitrile-0.085% trifluoroacetic acid, 35% acetonitrile)using a Model 172μ preparative HPLC system (Applied Biosystems) tofractionate four kinds of peptides. Sequences of the peptides obtainedwere determined by a protein sequencer.

-   T-19.4: GVETVDVSSTSR (12 residues) (SEQ ID NO: 29)-   T-21.0: TLDHFADR (8 residues) (SEQ ID NO: 30)-   T-33.4: FFVLR (5 residues) (SEQ ID NO: 31)-   T-47.1: AFPLEQFDLMPVTTIK (16 residues) (SEQ ID NO: 32)

Example 15 Isolation of PDT Gene

Of the N-terminal amino acid sequence (SEQ ID NO: 28) determined inExample 14, the following 5′ side primers were synthesized based onresidues 4-9 of the amino acid sequence (PDTN-4,5,6).

-   PDTN-4: TCYGCNGGNGAYGCNGG (SEQ ID NO: 33)-   PDTN-5: TCRGCNGGNGAYGCNGG (SEQ ID NO: 34)-   PDTN-6: AGYGCNGGNGAYGCNGG (SEQ ID NO: 35)

Further, the following 3′ side primers were synthesized based onresidues 5-11 of the amino acid sequence of SEQ ID NO: 32.

-   PDTC-3: GGCATNARRTCRAAYTGYTC (SEQ ID NO: 36)

Using the abovementioned primers, PCR was carried out in combinations ofPDTN-4×PDTC-3, PDTN-5×PDTC-3, and PDTN-6×PDTC-3. The PCR reaction wascarried out with KOD Dash (Toyobo Co., Ltd.) as a template using aPERKIN ELMER GeneAmp PCR System 9700. A reaction solution contained 1 μl(equivalent to 1 μg) of the genomic DNA of the PF1022 strain (FERMBP-2671) prepared by the method described in Example 5, 5 μl of 10-foldconcentrated reaction buffer attached to the enzyme, 5 μl of a 2 mM DNTPsolution, 1 μl each of the abovementioned primers prepared at aconcentration of 100 pmol/μl, 5 μl of dimethyl sulfoxide (specialreagent grade, Wako Pure Chemical Industries, Ltd.), and 1 μl of KODDash, and. 31 μl of sterile water was added to make a total volume of 50μl. The reaction was carried out by repeating 30 cycles of 30 seconds at94° C., 30 seconds at 55° C., and 30 seconds at 74° C., afterpretreatment at 94° C. for 5 minutes. The resulting reaction product wassubjected to agarose gel electrophoresis for analysis. The result showedthat a DNA fragment of approximately 200 bp was specifically amplifiedin combination of PDTN-6×PDTC-3.

Next, this DNA fragment of approximately 200 bp was excised from theagarose gel, extracted, purified, and then cloned using a TOPO TAcloning kit (Invitrogen). An inserted fragment of the resulting plasmidwas analyzed for the base sequence using a fluorescent DNA sequencer ABIPRISM 310 Genetic Analyzer (Perkin-Elmer). As a result, it was revealedthat the fragment encoded a sequence which partly agreed with the aminoacid sequence determined from the N terminal and peptide, showing theresulting DNA fragment was a part of the PDT gene.

Accordingly, screening of the genomic library of the substancePF1022-producing microorganism prepared in Example 5 was carried outwith this DNA fragment as a probe using an AlkPhos Direct Labelling andDetection System (Amersham Pharmacia Biotech). A phage DNA was extractedfrom the resulting positive clone and analyzed with restriction enzymes.As a result, it was revealed that an approximately 8.2-kbp SacI fragmentwas present as a DNA fragment to be hybridized with the abovementionedprobe. Accordingly, this fragment was subcloned into pUC118 to obtainplasmid pUC-PDT. By using this plasmid, the nucleotide sequence wasanalyzed using an ABIPRISM 377 sequencer (Applied Biosystems). FIG. 17shows the restriction enzyme map of the SacI fragment and the locationof the PDT gene. The presence of two introns in the PDT gene wassuggested by the nucleotide sequence analysis and was confirmed by thecDNA analysis. The nucleotide sequence of the PDT gene and the aminoacid sequence deduced from this sequence are shown in SEQ ID NO: 37 andSEQ ID NO: 38, respectively. Of the nucleotide sequence in SEQ ID NO:37, the base sequences 91-192 and 254-380 are introns.

Example 16 Construction of Plasmid for Disrupting PDT Gene of SubstancePF1022-Producing Microorganism

Plasmid pDPDT for disrupting the PDT gene was constructed as follows.

First, PCR was carried out using a commercially-available plasmidpUCSV-BSD (Funakoshi) containing a blasticidin S resistance gene (BSD)as a template and the oligonucleotides depicted in SEQ ID NO: 39 and SEQID NO: 40 as primers according to the method described in Example 2.After the reaction, a portion of the reaction solution was subjected toagarose gel electrophoresis, which confirmed that a DNA fragment ofapproximately 0.4 kbp was specifically amplified. Then, the remainingreaction solution was extracted with phenol:chloroform:isoamyl alcohol(25:24:1) and precipitated with ethanol. The precipitate was redissolvedin sterile water, and 60 μl of the resulting solution were digested withrestriction enzymes ClaI and BglII, after which agarose gelelectrophoresis was carried out, and a band of approximately 0.4 kbp wascut out according to an ordinary method to recover the DNA fragment.

Next, plasmid pDHBAR (Watanabe, M. et al., Appl. Environ. Microbiol.,65, 1036-1044 (1999)) was digested with restriction enzymes ClaI andBamHI, after which agarose gel electrophoresis was carried out and aband of approximately 2 kbp was cut out according to an ordinary methodto recover the DNA fragment. This DNA fragment and the abovementionedfragment of approximately 0.4 kbp were ligated to obtain plasmid pDHBSD.This plasmid was digested with restriction enzyme XbaI and the terminalswere blunted using a DNA Blunting Kit (Takara Shuzo Co., Ltd.), afterwhich agarose gel electrophoresis was carried out and a band ofapproximately 2.4 kbp was cut out according to an ordinary method torecover the DNA fragment.

Next, the plasmid pUC-PDT described in Example 15 was partially digestedwith restriction enzyme EcoRV, after which a DNA fragment ofapproximately 11.1 kbp was cut out according to an ordinary method torecover the DNA fragment. This DNA fragment and the above-mentionedfragment of approximately 2.4 kbp were ligated to obtain plasmid pDPDT(FIG. 18).

Example 17 Disruption of PDT Gene of Substance PF1022-ProducingMicroorganism

The plasmid pDPDT described in Example 16 was digested with restrictionenzyme SacI, and then dissolved in a TE buffer solution (10 mM Tris-HCl(pH 8.0), 1 mM EDTA) to make a final concentration of 1 μg/μl. StrainTF-57 described in Example 10 was transformed with this DNA solution bythe method described in Example 9. In this case, 100 μg/ml blasticidin Swas used as a selectable drug for transformants instead of hygromycin B.

About 100 strains of the resulting transformants were each culturedunder the conditions described in Example 5, centrifuged, collected andthen suspended in a buffer solution for disruption (50 mM Tris-HCl (pH8.0), 5 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol). This suspension wassubjected to ultrasound treatment, centrifuged to recover supernatant toobtain the cell extract. PDT activity was measured for this cell extractby the method described in Example 13. As a result, it was revealed thatno PDT activity was detected in 5 strains and these strains lacked thePDT activity.

Of these strains, strain TF-45 was selected and cultured along with aparent strain, TF-57, by the method described in Example 11 to detectPF1022-220. The result is shown in FIG. 19. PF1022-220 was detected atthe position of 20.303 minutes for strain TF-57 and 20.294 minutes forstrain TF-45. The peak area was about 4 times larger in strain TF-45than strain TF-57. From the result above, it was revealed thatPF1022-220 productivity was improved by impairing the PDT activity.

1. An isolated transformant producing a substance PF1022 derivative,said transformant is produced by introducing genes involved in abiosynthetic pathway from chorismic acid to p-aminophenylpyruvic acidinto a phenylalanine auxotrophic host induced from an organism thatproduces substance PF1022([cyclo(D-lactyl-L-N-methylleucyl-D-3phenyllactyl-L-N-methylleucyl)-D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl)])represented by formula (I):

wherein said genes comprise: (1) a gene encoding4-amino-4-deoxychorismate synthase, which gene comprises the DNAsequence encoding the amino acid sequence of SEQ ID NO: 2; (2) a geneencoding 4-amino-4-deoxychorismate mutase, which gene comprises the DNAsequence encoding the amino acid sequence of SEQ ID NO: 4; and (3) agene encoding 4-amino-4-deoxyprephenate dehydrogenase, which genecomprises the DNA sequence encoding the amino acid sequence of SEQ IDNO: 6, and wherein the substance PF1022 derivative is a compound offormula (III)([cyclo(D-lactyl-L-N-methylleucyl-D-3-(4-nitrophenyl)lactyl-L-N-methylleucyl-D-lactyl-L-N-methylleucyl-D-3-(4-nitrophenyl)lactyl-L-N-methylleucyl)])represented by the following formula:

or a compound of formula (V)([cyclo(D-lactyl-L-N-methylleucyl-D-3-(4-aminophenyl)lactyl-L-N-methylleucyl-D-lactyl-L-N-methylleucyl-D-3-(4-aminophenyl)lactyl-L-N-methylleucyl)]) represented by the following formula:

wherein the phenylalanine auxotrophic host is obtained from Myceliasterilia by disrupting an endogenous chorismate mutase gene encoding theamino acid sequence of SEQ ID NO: 27 and/or a prephenate dehydratasegene encoding the amino acid sequence of SEQ ID NO: 38 by homologousrecombination, “so as to reduce endogenous chorismate mutase and/orprephenate degydratase activity” wherein the Mycelia sterilia is thestrain deposited with the National Institute of Advanced IndustrialScience and Technology under an accession number of FERM BP-2671.
 2. Thetransformant according to claim 1, wherein the phenylalanine auxotrophichost lacks endogenous chorismate mutase activity and/or prephenatedehydratase activity.
 3. The transformant according to claim 1, whereinat least one of the genes is a gene from genus Streptomyces.
 4. Thetransformant according to claim 1, wherein the gene encoding4-amino-4-deoxychorismate synthase comprises the DNA sequence of SEQ IDNO:
 1. 5. The transformant according to claim 1, wherein the geneencoding 4-amino-4-deoxychorismate mutase comprises the DNA sequence ofSEQ ID NO:
 3. 6. The transformant according to claim 1, wherein the geneencoding 4-amino-4-deoxyprephenate dehydrogenase comprises the DNAsequence of SEQ ID NO:
 5. 7. The transformant according to claim 1,wherein the gene encoding 4-amino-4-deoxychorismate synthase, the geneencoding 4-amino-4-deoxychorismate mutase, and the gene encoding4-amino-4-deoxyprephenate dehydrogenase comprise the DNA sequence of SEQID NO: 1, the DNA sequence of SEQ ID NO: 3, and the DNA sequence of SEQID NO: 5, respectively.
 8. A method for producing a substance PF1022derivative, which comprises the steps of culturing the transformant ofclaim 1 and collecting a substance PF1022 derivative.
 9. The method forproducing a substance PF1022 derivative according to claim 8, whereinthe substance PF1022 derivative is a compound of formula (III):

or a compound of formula (V):